The SMN complex, an assemblyosome of ribonucleoproteins

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Spinal muscular atrophy is a common, often lethal,neurodegenerative disease that results from low levels of, orloss-of-function mutations in, the SMN (survival of motorneurons) protein. SMN oligomerizes and forms a stablecomplex with five additional proteins: Gemins 2–6. SMN alsointeracts with several additional proteins referred to as‘substrates’. Most of these substrates contain a domainenriched in arginine and glycine residues (the RG-rich domain),and are constituents of different ribonucleoprotein complexes.Recent studies revealed that the substrates can be modified by an arginine methyltransferase complex, the methylosome.This forms symmetrical dimethylarginines within the RG-richdomains of the substrates, thereby converting them to high-affinity binders of the SMN complex, and most likely providingregulation of the ribonucleoprotein assembly processes.

AddressesHoward Hughes Medical Institute, and the Department of Biochemistryand Biophysics, University of Pennsylvania School of Medicine,Philadelphia, PA 19104-6148, USA*e-mail: [email protected]

Current Opinion in Cell Biology 2002, 14:305–312

0955-0674/02/$ — see front matter© 2002 Elsevier Science Ltd. All rights reserved.

AbbreviationspolII RNA polymerase IIRNP ribonucleoprotein SMA spinal muscular atrophySMN survival of motor neuronssnRNP small nuclear RNPsnoRNP small nucleolar RNPUsnRNP uridine-rich snRNP

IntroductionSpinal muscular atrophy (SMA) is an autosomal recessiveneurodegenerative disorder. It is the leading genetic causeof infant mortality [1] and is characterized by the degener-ation of α-motor neurons in the spinal cord, resulting inmuscular atrophy and ultimately paralysis [2]. Using acombination of genetic analysis and physical mapping inpatients suffering from SMA, the gene responsible for thecondition has been identified as SMN (survival of motorneurons) [3]. In humans, this gene is duplicated as aninverted repeat on chromosome 5 (in the 5q13 region) [3],and only deletions or loss-of-function mutations in thetelomeric copy, SMN1, give rise to SMA (reviewed in [4]).The centromeric copy, SMN2, mostly expresses a functionallydefective form of the protein that lacks the carboxyl terminusencoded by exon 7.

SMN is an essential gene in divergent organisms, includinghuman, mouse, chicken, Caenorhabditis elegans and

Schizosaccharomyces pombe [3,5–10], indicating that itprovides a function required by all cells. In this review, weoutline the properties of the SMN complex as an assembly-osome for many different classes of ribonucleoprotein(RNP) complexes.

SMN is found in the cytoplasm and in nuclear gemsThe SMN protein is ubiquitously expressed in all tissuesof metazoan organisms [3]. At the subcellular level, SMNis found in the cytoplasm and the nucleus, where it ispresent throughout the nucleoplasm and is highly concentrated in distinct subcellular structures called‘gems’ [11] — nuclear bodies similar in size and number toCajal (coiled) bodies. These latter structures were firstidentified at the turn of the 20th century and are known tocontain high levels of small nuclear RNPs (snRNPs) andsmall nucleolar ribonucleoproteins (snoRNPs) [12,13].Although Cajal bodies have been implicated insnRNP/snoRNP biogenesis, trafficking and regeneration,as well as in histone mRNA 3′ processing, their precisefunction still awaits clarification. Cells devoid of Cajal bodiesare both viable and competent to divide [12–15].

Double-label immunofluorescence studies were carriedout, using antibodies against SMN as a gem marker, andp80 coilin as a Cajal body marker [16]. These studiesdemonstrated that gems and Cajal bodies are separateentities in fetal tissues and certain types of cultured cells(for example, HeLa PV cells); whereas in adult tissues andcertain other cell lines (for example, HeLa S3 cells) thesebodies predominantly co-localize [11,17–19]. Gems andCajal bodies should therefore be viewed as distinct andindependent nuclear structures that have a dynamicfunctional relationship and are often associated [17,20,21].

Gems are also distinct from interchromatin granule clusters(also known as ‘speckles’), which are DNA-free nucleardomains composed of tightly packed ribonucleoproteinparticles that accumulate mature snRNPs and proteinsplicing factors [14]. Although gems contain a particularlyhigh concentration of the components of the SMN complex,their specific function, if any, is not yet known.

SMN is part of a multiprotein complexThe SMN protein is a 294-amino-acid polypeptide thatoligomerizes and forms a complex with at least fiveadditional proteins: Gemin2 (formerly known as SIP1),Gemin3/dp103 (a DEAD-box RNA helicase), Gemin4,Gemin5 (a WD-repeat protein) and Gemin6 [22–27].These proteins are integral components of the SMNcomplex — immunoprecipitation studies using anti-SMN

The SMN complex, an assemblyosome of ribonucleoproteinsSergey Paushkin, Amélie K Gubitz, Séverine Massenet andGideon Dreyfuss*

antibodies or antibodies to the Gemins lead to theirco-purification, even under stringent (high salt) conditions(Figure 1a). Gemins 2, 3 and 5 interact directly withSMN, while Gemin4 associates indirectly, via strongbinding to Gemin3 [22–24,26] (Figure 1b). The exactnature of the association between Gemin6 and the complexis presently unknown.

In addition to the tight interaction with its complexcomponents, SMN also interacts directly, but perhaps lessavidly or more transiently, or both, with several otherproteins that serve as substrates (see below). Importantly,all integral components of the SMN complex characterizedso far show an overlapping subcellular localization pattern and also have common substrates for proteininteraction: the Sm proteins. A set of seven Sm proteins orSm-like (Lsm) proteins are associated with uridine-richsmall nuclear RNAs (UsnRNAs), together with severaluridine-rich small nuclear ribonucleoprotein (UsnRNP)-specific proteins, to form UsnRNP particles. Theseparticles are crucial components of the spliceosomalmachinery [23,24,26–28].

Apart from the DEAD-box motif in Gemin3, the onlyother notable structural motif found in the Gemins is thepresence of 13 WD-repeat domains in Gemin5, suggestingthe capacity for multiple protein–protein interactions[26,29,30••]. It is therefore conceivable that Gemin5 servesas a platform for protein assembly and engages in multiplesubstrate interactions such as binding to Sm proteins.

Substrates of the SMN complexBesides the major integral components of the SMN particle(i.e. the Gemins), which are stably associated, severaladditional proteins can be co-purified. These are transientlyassociated and are referred to as ‘SMN complex substrates’.It is intriguing that most SMN complex substrates characterized so far are constituents of various RNP com-plexes. What renders all these substrates recognisable bythe SMN complex? Domain mapping by direct-bindingexperiments and detailed comparison of the amino acidsequences of all known substrates revealed that most ofthem have a domain enriched in arginine and glycine residuesthat is required for interaction with SMN (Figure 2; Table 1).However, it should be noted that several additionalSMN-complex interaction partners have been reported thatneither contain RG-rich motifs nor interact with RNPs, such asZRP1, profilin, p53 and the FUSE-binding protein [31–34].

Deletion of the RG-rich domains in SMN complexsubstrates completely abolishes their ability to bind theSMN complex [28,35,36•,37]. Thus it appears that theSMN complex interacts with many, perhaps all, of itssubstrates using a common mechanism that involves aninteraction with specific RG motifs. Potentially, arginineresidues in the RG sequence can be methylated, and thismodification appears to be critically important for SMNbinding. Indeed, the association of SMN with Sm proteinsis mediated by a direct interaction with symmetricaldimethylarginine-modified RG domains of SmD1 andSmD3 [38••]. These symmetrical dimethylarginines are

306 Nucleus and gene expression

Figure 1

SMN is part of a multiprotein complex.(a) Immunoprecipitations using anti-SMN(2B1), anti-Sm (Y12), or control non-immune(SP2/0) antibodies from [35S]methionine-labeled total HeLa cell extract. Theimmunoprecipitated proteins were analysed bySDS–PAGE and autoradiography. The anti-SMN antibody 2B1 (lane 2) precipitates SMN,Gemins 2–5 (labeled in red; Gemin6 is onlyvisible after longer exposure), the Sm proteinsB/B′, D1–D3, E, F and G (labeled in green),and several not yet characterized/non-specificproteins. In contrast, the anti-Sm antibody Y12(lane 1) precipitates the major U1 snRNA-specific proteins U1 70K, U1A and U1C(labeled in black), the eight common Smproteins (labeled in green) but none of the corecomponents of the SMN complex. The SP2/0lane shows the background of theimmunoprecipitations (lane 3). (b) Schematicrepresentation of the SMN complex. Gemins 2,-3 and -5 bind directly to SMN, while Gemin4is associated through direct interaction withGemin3. The precise binding site for Gemin6so far has not been defined, and Gemin6 istherefore omitted from the model. To simplifythe model, the complex is depicted in dimericrather than oligomeric form.

Gemin4

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N)

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trol)

(a) (b)

Current Opinion in Cell Biology

formed by the 20S arginine methyltransferase complex,termed the methylosome, that contains the methyltransferaseJBP1 (PRMT5), pICln and MEP50 (a WD-repeat protein)[39,40••,41]. JBP1 binds SmD1 and SmD3 via their RGdomains, whereas pICln and MEP50 bind the Sm domains[41]. MEP50 interacts with JBP1 and a subset of Smproteins, in particular SmB and SmD2.

Because WD-repeat proteins provide a platform for multipleprotein interactions [29], MEP50 may function to mediatethe interaction of multiple substrates with the methylosome.This putative role of MEP50 as a protein-interactionplatform for the methylosome may be similar to thatenvisioned for Gemin5, also an Sm-binding protein, in theSMN complex [26]. Interestingly, all of the known compo-nents of the methylosome bind Sm proteins, including Smproteins that are not methylated (e.g. SmD2). This suggeststhat, in addition to producing properly arginine-dimethylatedsubstrates for the SMN complex, the methylosome may beinvolved in Sm protein rearrangements or in the pre-assemblyrequired for snRNP biogenesis — namely, that it plays a rolein the RNP assembly process beyond just the methylationof arginine residues [41].

SmB/B′ and Lsm4 also contain symmetrical dimethylarginineresidues in vivo that are necessary for SMN binding [42]. Itis conceivable that all RG-domain-containing substrates ofthe SMN complex undergo a pathway similar to thatdescribed for the Sm proteins [38••], and that the methy-losome prepares and directs them to the SMN complex byconverting them to high-affinity SMN binders [40••]. Thecritical role of methylation in the interaction of SMN withits complex substrates suggests that deficiencies in methy-lation may have harmful consequences similar to reductionin the level of, or mutations in, SMN, as seen in SMApatients. Hence, undermethylation of SMN substrates inmotor neurons may also be deleterious to these cells [38••].

The carboxy-terminal domain of SMN, spanning exons 6and 7, is essential for substrate binding, and its deletioncompletely disrupts the interaction of SMN with all of itsknown substrates [22,28,35,36•,37]. There are several linesof evidence to indicate the key role of the carboxy-terminaldomain of SMN in binding its substrates. First, the aminoacid sequence of the carboxy-terminal region of SMN ishighly conserved in divergent organisms [9]. Second, over96% of SMA patients show homozygous absence of either

SMN1 in exons 7 and 8 or exon 7 alone [43], implying thatthis domain is critical for the physiological functions ofSMN. Third, deletion of the carboxyl terminus abolishesthe ability of SMN to oligomerize [28,44]. This oligomer-ization defect results in the inability of these mutants toform large SMN particles (see below).

Recently, the importance of the SMN exon-3-encodedTudor domain for substrate binding has been emphasized,particularly with respect to the Sm proteins [42,45–47]. Inour hands, however, deletion of the Tudor domain does notinfluence the ability of SMN to interact with its substrates(J Baccon et al., personal communication). Additionalexperiments, including, eventually, determination of the

The SMN complex, an assemblyosome of ribonucleoproteins Dreyfuss et al. 307

Figure 2

Schematic representation of arginine- and glycine-rich domains of theSMN complex substrates. RG-rich domains are typed in yellow withblack background.

GSKKSNKNKSGKNQFNRGGGHRGRGGLNMRGGNFRGGAPGNRGGYNRRGNMPQRGGGGGGSGGIGYPYPRAPVFPGRGSYSNRGNYNRGGMPNRGNYNQNFRGRGNNRGYKNQSQGYNQWQQGQFWGQKPWSQHYHQGYY

hnRNP Uc188

SmB c139

ARVPLAGAAGGPGIGRAAGRGIPAGVPMPQAPAGLAGPVRGVGGPSQQVMTPQGRGTVAAAAAAATASIAGAPTQYPPGRGGPPPPMGRGAPPPGMMGPPPGMRPPMGPPMGIPPGRGTPMGMPPPGMRPPPPGMRGLL

SmD1 c29

KREAVAGRGRGRGRGRGRGRGRGRGGPRR

SmD3 c32

AGRGKAAILKAQVAARGRGRGMGRGNIFQKRR

Lsm4 c54

KGRGRGGLQQQKQQKGRGMGGAGRGVFGGRGRGGIPGTGRGQPEKKPGRQAGKQ

Fibrillarin n81

MKPGFSPRGGGFGGRGGFGDRGGRGGRGGFGGGRGRGGGFRGRGRGGGGGGGGGGGGGRGGGGFHSGGNRGRGRGGKRGNQ

RGRAGYSQRGG M APGSARG RG RGRGGAQQQ RG

hnRNP Q1518–549


Table 1

RG-rich-domain-containing substrates of the SMN complex.

Name of substrate RNP Function References

Sm proteins U snRNPs Pre-mRNA splicing [22,35]Sm-like (Lsm) proteins U6 snRNP Pre-mRNA splicing [35]Fibrillarin, Gar1 snoRNPs Ribosome biogenesis [37,46]Heterogeneous nuclear RNP U, Q Heterogeneous nuclear RNPs Pre-mRNA splicing [54,55]RNA helicase A Transcriptosome Transcription [36�]Coilin Unknown Association of SMN with Cajal bodies? [56]

atomic structures of SMN bound to its substrates, will beneeded to define precisely the binding sites.

The SMN complex is a large 50S particleSedimentation of HeLa cell cytoplasmic extracts on sucrosegradients reveals that SMN, Gemin2 and Gemin3 co-migratein a large particle with a peak at ~50S (Figure 3a; S Paushkinand G Dreyfuss, unpublished data). This native complex issomewhat heterodisperse since SMN-containing material isfound in a broad range, between 40S and 70S. The SMN par-ticle is resistant to RNaseA, high salt and EDTA, indicatingthat it is not associated with ribosomal subunits and representsa novel, authentic particle containing all the known compo-nents of the SMN complex. Furthermore, its relatively broadsedimentation profile in sucrose gradients implies that theSMN particle exhibits a remarkable degree of flexibility inits assembly properties. A 40S–70S size for the cytoplasmicSMN complex is much larger than has been reported previ-ously, including the observation that SMN is associated witha 20S complex in nuclear extracts of HeLa cells [48]. It ispossible that this discrepancy results from differences inexperimental conditions or from the cellular fractions used.

Most SmB (and other Sm proteins) is found in fractionscorresponding to 2S–20S (Figure 3a), suggesting that, at anygiven time, only a small amount of Sm proteins is associatedwith the SMN complex.

Most of the mass of the 50S SMN particle comprises SMNand Gemin2, and these two proteins are essential for itsformation. Co-expression of SMN and Gemin2, but not ofeither protein alone, is sufficient to form a particle of a size(40S) approaching that of the native SMN particle(Figure 3b). It is likely that the other core components ofthe SMN complex, such as Gemins 3–6, make up the sizedifference and are required for formation of the native 50SSMN particle. Indeed, analysis of the purifiedSMN–Gemin2 complex by SDS–PAGE reveals that, asidefrom SMN and Gemin2, several additional components ofthe SMN complex, including Gemin3, -4 and -5, are present(S Paushkin and G Dreyfuss, unpublished data). However,the amount of SMN and Gemin2 is far greater than that ofany of the other proteins, strongly suggesting that the coreof the native SMN particle has a simple protein compositioncomprising only two proteins, SMN and Gemin2.


Figure 3

The SMN complex is a large 50S particle.(a) The native SMN complex sediments as a50S particle. Cytoplasmic extracts wereprepared from HeLa cells and sedimented ona 5–20% sucrose gradient. Fractions werecollected (indicated by numbers 1–20),separated by SDS–PAGE and Westernblotted using anti-SMN, anti-Gemin2,anti Gemin3 and anti-Sm (Y12) monoclonalantibodies to detect the proteins indicated.(b,c) The SMN Y272→C point mutant isdeficient in particle formation. Myc–SMN (b) orMyc–SMN Y272→C (c) were expressed in293 cells together with flag–Gemin2.Cytoplasmic extracts were prepared andincubated with immobilized anti-flag antibody.Bound proteins were eluted with flag peptideand sedimented on 5–20% sucrose gradients.Fractions were collected (indicated bynumbers), separated by SDS–PAGE, andWestern blotted using the appropriateantibodies to detect the proteins indicated.

tota

l1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

SMN

Gemin2

Gemin3

SMN

Gemin2

Gemin2

SMN Y272→C

SmB

(b)

(c)

70S50S(a) 6S


The role of Gemin2 in the formation of the SMN particleis particularly intriguing because, like a chaperone, it iscritical for proper oligomerization of SMN (S Paushkin andG Dreyfuss, unpublished data). In its simplicity of compo-sition, the SMN particle is reminiscent of the particles ofthe chaperonins GroEL and GroES [49].

As described above, the evolutionary conserved carboxy-terminal domain of SMN is required for its self-associationand binding to its substrates. Mutations in this domain are fre-quently found in non-SMN1-deleted SMA patients, includingthe deletion of exon 7 (SMN∆Ex7) and the most commonpoint mutation Y272→C [43]. Remarkably, SMN∆Ex7 andY272→C mutant proteins are not able to form 40S particles(Figure 3c; S Paushkin and G Dreyfuss, unpublished data).This demonstrates that the carboxy-terminal region of SMN

is necessary for SMN particle formation. These observationsindicate a functional link between SMN particle formationand SMN function, because the carboxy-terminal mutants arenot only functionally deficient but are also defective in theformation of the SMN particle.. It can therefore be concludedthat the SMN particle is the native functional entity, and itsformation is essential for the biological activities of SMN.

Functions of the SMN complex inribonucleoprotein assemblyMost of the known substrates of the SMN complex areconstituents of various types of RNP complexes that playdiverse roles in many aspects of cellular RNA metabolism.This observation implies that the SMN complex is alsoinvolved in many aspects of RNA metabolism. Indeed,studies in Xenopus laevis oocytes and mammalian cells


Figure 4

Schematic representation of UsnRNPbiogenesis. The uridine-rich small nuclear RNAs(UsnRNAs), with the exception of U6, aretranscribed by RNA polymerase II (polII) asprecursors that contain additional nucleotides atthe 3′ end and a monomethylated m7GpppG(m7G) cap structure at the 5′ end. This capstructure is recognised by the nuclear-cap-binding complex (CBC), a heterodimericcomplex composed of CBP20 and CBP80,both required for binding to the m7G-capstructure. The adaptor protein Phax(phosphorylated adaptor for RNA export) bindsboth CBC and UsnRNAs, and mediates theirinteraction with the export receptorCRM1/Exportin1 (Xpo1). CRM1, together withRan–GTP, exports newly transcribed UsnRNAsinto the cytoplasm. Following export to thecytoplasm, GTP hydrolysis of Ran anddephosphorylation of Phax, UsnRNAs bind tothe SMN complex, which contains Sm proteins.SmD1, SmD3 and perhaps SmB are firstmethylated by the methylosome, whichproduces symmetrical dimethylarginines anddirects them to the SMN complex. Most likely,the SMN complex brings together theSm proteins and UsnRNAs by binding both. Thisresults in generation of the UsnRNP particles.This step is required for cap hypermethylationand 3′-end maturation. A properly assembledSm core and the m3G-cap structure areprerequisite for UsnRNP import into thenucleus. The m3G-cap structure is specificallybound by snurportin, which then interacts withthe import receptor importin β and, togetherwith a not yet identified import receptor thatrecognises the Sm core, mediates UsnRNAimport. The SMN complex is not released fromthe UsnRNP after Sm core assembly and ispresent during and after cap hypermethylationand also remains bound to the UsnRNPs beforetheir import. Thus, the SMN complex not onlyplays a role in snRNP core assembly; it is alsoimplicated in the entire snRNP core biogenesisin the cytoplasm (reviewed in [57]). CB, Cajalbody; NPC, nuclear pore complex. TMG, m3G.

E

BD1

D3

D3

D1B

B D3

D2E

D1

pre-UsnRNA8080

20

20

Phax

CBC

UsnRNAexport

m7G

m7G

UsnRNPassembly

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SMNcomplex

Snurportin

plCln

Methylationof Sm proteins

Caphypermethylation

Methylosome

Importreceptor

FG

B D3

D2E

D1

FG

B D3

D2

D2

E

D1

F

F

G

G

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pol II

NUCLEUS

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Gemin4Gemin3

Gemin5

Gemin2SMN

TMG

MEP50 JBP1 (PRMT5)


Gem

reveal an active role of the SMN complex in the assemblyof UsnRNPs [21,30••,45,50]. UsnRNPs, together withsplicing factors, comprise the macromolecular machinerythat catalyzes pre-mRNA splicing. The formation ofUsnRNPs takes place in the cytoplasm and involves theinteraction of newly exported UsnRNA with seven Smproteins (Figure 4). SMN complex interaction directlywith Sm proteins (see above) and with UsnRNAs [51] isrequired to mediate the assembly of UsnRNPs both inXenopus oocytes and in Xenopus egg extracts [30••,50].

The SMN complex participates in all steps of UsnRNPassembly, and it chaperones UsnRNPs throughout theentire process of their biogenesis in the cytoplasm. It doesso by being associated with newly exported UsnRNAs,assembled Sm cores, m3G (m2,2,7GpppG)-cappedUsnRNAs and finally with the pre-import complex con-taining m3G-capped snRNP cores bound to snurportin(S Massenet and G Dreyfuss, unpublished data). Thus, theassembly of UsnRNPs is a complex and intricate processmediated by both the SMN particle and the methylosomethat modifies and targets the Sm proteins to the SMNparticle for the assembly into UsnRNPs. Our view of therole of the SMN particle and the methylosome in theprocess of the UsnRNP assembly is illustrated in Figure 4.

A dominant-negative mutant of SMN, SMN∆N27, inhibitspre-mRNA splicing in vitro [21]. This mutant also causes adramatic re-organization of UsnRNPs and the RNApolymerase II (polII) transcription/processing complexmachinery in the nucleus, most likely by altering the regen-eration of functionally active UsnRNPs or other componentsof the spliceosome and the components of the polII tran-scription machinery [21,36•]. The involvement of SMN inthe organization/assembly of polII transcription complexesis based on its ability to interact with RNA helicase A(RHA). This protein is a DEAD-box RNA helicase thatbinds polII and reportedly functions in transcription [52,53].

Finally, the SMN complex interacts with GAR1 and fibrillar-in, specific markers of the two classes of snoRNPs that areinvolved in the processing and modification of ribosomalRNA [37,46]. Thus, the SMN complex may also play a rolein the metabolism of snoRNPs.

ConclusionsThe SMN complex operates as a macromolecular assemblymachine (an assemblyosome) in a myriad fundamental cellu-lar events, including transcription and the processing of mostpre-mRNAs and ribosomal RNAs. The need for such anassembly machine came as a surprise because several RNPshave been known to assemble in vitro without the aid of theSMN complex. However, within the intricate microenviron-ment of the cell, such assembly processes are probably tooslow and inefficient without the assistance of the SMN par-ticle. The methylosome, and possibly other protein argininemethyltransferases, is likely to provide regulation of theRNP-assembly processes that the SMN particle mediates.

Further understanding of the structure and function ofthe SMN particle and the methylosome will help tounveil the detailed mechanisms of their activity, as wellas the molecular pathogenesis of SMA.

AcknowledgementsWe are grateful to members of our laboratory, in particular ZissimosMourelatos, Tracey Golembe and Jennifer Baccon for helpful discussionsand comments on the manuscript. This work was supported by theAssociation Française contre les Myopathies (AFM) and through a grantfrom the National Institute of Health. G Dreyfuss is an Investigator of theHoward Hughes Medical Institute, USA.

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28. Pellizzoni L, Charroux B, Dreyfuss G: SMN mutants of spinalmuscular atrophy patients are defective in binding to snRNPproteins. Proc Natl Acad Sci USA 1999, 96:11167-11172.

29. Smith TF, Gaitatzes C, Saxena K, Neer EJ: The WD repeat: acommon architecture for diverse functions. Trends Biochem Sci1999, 24:181-185.

30. Meister G, Buhler D, Pillai R, Lottspeich F, Fischer U: A multiprotein •• complex mediates the ATP-dependent assembly of spliceosomal

U snRNPs. Nat Cell Biol 2001, 3:945-949.Analysis of U1 small nuclear ribonucleoprotein (snRNP) assembly by nativegel electrophoresis using Xenopus laevis egg extract and in vitro transcribedand radioactively labeled U1 snRNA. This cell-free assay system revealsthat the assembly of U1 snRNP is dependent upon ATP. Furthermore,depletion of the SMN–Gemin2-containing complex from the extract abolishesthe assembly, despite the presence of high levels of Sm proteins. Additionof an affinity-purified macromolecular SMN complex to the immunodepletedextract restores the U1 snRNP assembly, thus demonstrating that the SMNcomplex mediates uridine-rich snRNP assembly. The authors also show thatthe affinity-purified macromolecular SMN complex contains at least16 components.

31. Giesemann T, Rathke-Hartlieb S, Rothkegel M, Bartsch JW,Buchmeier S, Jockusch BM, Jockusch H: A role for polyprolinemotifs in the spinal muscular atrophy protein SMN. Profilins bindto and colocalize with SMN in nuclear gems. J Biol Chem 1999,274:37908-37914.

32. Williams BY, Hamilton SL, Sarkar HK: The survival motor neuronprotein interacts with the transactivator FUSE binding proteinfrom human fetal brain. FEBS Lett 2000, 470:207-210.

33. Gangwani L, Mikrut M, Theroux S, Sharma M, Davis RJ: Spinalmuscular atrophy disrupts the interaction of ZPR1 with the SMNprotein. Nat Cell Biol 2001, 3:376-383.

34. Young PJ, Day PM, Zhou J, Androphy EJ, Morris GE, Lorson CL: Adirect interaction between the survival motor neuron protein andp53 and its relationship to spinal muscular atrophy. J Biol Chem2002, 277:2852-2859.

35. Friesen WJ, Dreyfuss G: Specific sequences of the Sm and Sm-like(Lsm) proteins mediate their interaction with the spinal muscularatrophy disease gene product (SMN). J Biol Chem 2000,275:26370-26375.

36. Pellizzoni L, Charroux B, Rappsilber J, Mann M, Dreyfuss G: A • functional interaction between the survival motor neuron complex

and RNA polymerase II. J Cell Biol 2001, 152:75-85.The authors show that SMN interacts with RNA helicase A (RHA) in vitro,and that this interaction is impaired in SMN mutants found in patients withspinal muscular atrophy. The RG-rich domain of RHA, which is neitherrequired for its interaction with RNA polymerase II nor with transcriptionalactivators, is necessary for the interaction with SMN. Co-immunoprecipitationexperiments demonstrate that the SMN complex is associated with RHA andRNA polymerase II in vivo. Transfection of cells with a dominant-negativemutant of SMN, SMN∆N27, causes accumulation of RNA polymerase II andRHA in nuclear aggregates that contain the known markers of gems andCajal bodies, and inhibits RNA polymerase I and II transcription in vivo.These findings suggest a role for the SMN complex in the assembly of theRNA polymerase II transcription/processing machinery.

37. Pellizzoni L, Baccon J, Charroux B, Dreyfuss G: The survival of motorneurons (SMN) protein interacts with the snoRNP proteinsfibrillarin and GAR1. Curr Biol 2001, 11:1079-1088.

38. Friesen WJ, Massenet S, Paushkin S, Wyce A, Dreyfuss G: SMN, the •• product of the spinal muscular atrophy gene, binds preferentially

to dimethylarginine-containing protein targets. Mol Cell 2001,7:1111-1117.

This is a demonstration that methylation of arginine residues is a novelmechanism to promote specific protein–protein interactions. Bacteriallyexpressed SmD1 and SmD3 that do not contain the symmetrically dimethy-lated arginines (sDMAs) found in the RG-rich domains of their mammalianorthologues are not able to bind SMN from HeLa cells. This indicates thatsDMA post-translational modifications may be important for Sm proteinassociation with the SMN complex. This hypothesis is tested using peptidescorresponding to the carboxy-terminal 29 and 32 amino acids of SmD1 andSmD3, respectively, with or without the specific sDMA modifications formedin vivo. Because only the symmetrically methylated RG peptides are foundto be capable of binding to SMN, it can be concluded that the native SMNcomplex binds preferentially to symmetrically dimethylated SmD1 and SmD3in cell extracts.

39. Meister G, Eggert C, Buhler D, Brahms H, Kambach C, Fischer U:Methylation of Sm proteins by a complex containing PRMT5 andthe putative U snRNP assembly factor pICln. Curr Biol 2001,11:1990-1994.

40. Friesen WJ, Paushkin S, Wyce A, Massenet S, Pesiridis GS, •• Van Duyne G, Rappsilber J, Mann M, Dreyfuss G: The methylosome,

a 20S complex containing JBP1 and pICln, producesdimethylarginine-modified Sm proteins. Mol Cell Biol 2001,21:8289-8300.

This work demonstrates that SmD1 and SmD3 are in a complex with themethyltransferase JBP1, which produces symmetrical dimethylarginines inthe RG-rich domains of both proteins. The existence of two distinct SmD1-and SmD3-containing complexes, sedimenting at 6S and 20S on sucrosegradients, is shown. The 6S complex contains pICln, SmD1 and SmD3,whereas the 20S complex, termed the ‘methylosome’, contains these threeproteins together with the methyltransferase JBP1. The study also demon-strates that SmD3 from the methylosome, but not from the 6S complex, canbe transferred to the SMN complex, suggesting that the methylation of Smproteins by the methylosome directs them to the SMN complex.

41.Friesen WJ, Wyce A, Paushkin S, Abel L, Rappsilber J, Mann M,Dreyfuss G: A novel WD repeat protein component of themethylosome binds Sm proteins. J Biol Chem 2002,277:8243-8247.

42. Brahms H, Meheus L, de Brabandere V, Fischer U, Luhrmann R:Symmetrical dimethylation of arginine residues in spliceosomalSm protein B/B′′ and the Sm-like protein Lsm4, and theirinteraction with the SMN protein. RNA 2001, 7:1531-1542.

43. Wirth B: An update of the mutation spectrum of the survival motorneuron gene (SMN1) in autosomal recessive spinal muscularatrophy (SMA). Hum Mutat 2000, 15:228-237.

44. Lorson CL, Strasswimmer J, Yao JM, Baleja JD, Hahnen E, Wirth B,Le T, Burghes AH, Androphy EJ: SMN oligomerization defectcorrelates with spinal muscular atrophy severity. Nat Genet 1998,19:63-66.


45. Buhler D, Raker V, Luhrmann R, Fischer U: Essential role for thetudor domain of SMN in spliceosomal U snRNP assembly:implications for spinal muscular atrophy. Hum Mol Genet 1999,8:2351-2357.

46. Jones KW, Gorzynski K, Hales CM, Fischer U, Badbanchi F,Terns RM, Terns MP: Direct interaction of the spinal muscularatrophy disease protein SMN with the small nucleolar RNA-associated protein fibrillarin. J Biol Chem 2001,276:38645-38651.

47. Selenko P, Sprangers R, Stier G, Buhler D, Fischer U, Sattler M: SMNTudor domain structure and its interaction with the Sm proteins.Nat Struct Biol 2001, 8:27-31.

48. Meister G, Buhler D, Laggerbauer B, Zobawa M, Lottspeich F,Fischer U: Characterization of a nuclear 20S complex containingthe survival of motor neurons (SMN) protein and a specificsubset of spliceosomal Sm proteins. Hum Mol Genet 2000,9:1977-1986.

49. Ranson NA, White HE, Saibil HR: Chaperonins. Biochem J 1998,333:233-242.

50. Fischer U, Liu Q, Dreyfuss G: The SMN–SIP1 complex has anessential role in spliceosomal snRNP biogenesis. Cell 1997,90:1023-1029.

51. Yong J, Pellizzoni L, Dreyfuss G: Sequence-specific interaction ofU1 snRNA with the SMN complex. EMBO J 2002, 21:1188-1196.

52. Nakajima T, Uchida C, Anderson SF, Lee CG, Hurwitz J, Parvin JD,Montminy M: RNA helicase A mediates association of CBP withRNA polymerase II. Cell 1997, 90:1107-1112.

53. Anderson SF, Schlegel BP, Nakajima T, Wolpin ES, Parvin JD: BRCA1protein is linked to the RNA polymerase II holoenzyme complexvia RNA helicase A. Nat Genet 1998, 19:254-256.

54. Mourelatos Z, Abel L, Yong J, Kataoka N, Dreyfuss G: SMN interactswith a novel family of hnRNP and spliceosomal proteins. EMBO J2001, 20:5443-5452.

55. Rossol W, Kroning AK, Ohndorf UM, Steegborn C, Jablonka S,Sendtner M: Specific interaction of SMN, the spinal muscularatrophy determining gene product, with hnRNP-R and gry-rbd/hnRNP-Q: a role for SMN in RNA processing in motor axons?Hum Mol Genet 2002, 11:93-105.

56. Hebert MD, Szymczyk PW, Shpargel KB, Matera AG: Coilin formsthe bridge between Cajal bodies and SMN, the spinal muscularatrophy protein. Genes Dev 2001, 15:2720-2729.

57. Will CL, Luhrmann R: Spliceosomal U snRNP biogenesis, structureand function. Curr Opin Cell Biol 2001, 13:290-301.


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The SMN complex, an assemblyosome of ribonucleoproteins